Unconjugated bilirubin, the hydrophobic end-product of heme degradation, is generally held to be little more than a metabolic by-product of heme catabolism. Heme oxygenase (HO) is the rate-limiting enzyme in the conversion of heme to bilirubin. The inducible isoform of this enzyme, HO-1, is up-regulated in response to inflammatory stimuli and has been shown to be cytoprotective in a number of tissue injury models. Hence, we speculate that bilirubin may be a primary mediator of the protective effects of HO-1 in the liver and in other organ systems. The proposed studies will specifically investigate the hypothesis that bilirubin functions as an endogenous inhibitor of the inflammatory response. The broad, long-term objectives of the outlined research proposal are to characterize the mechanism(s) underlying the anti-inflammatory properties of bilirubin and to explore potential therapeutic implications. The experiments outlined in Specific Aim 1 are designed to evaluate the potency and establish the pathways whereby bilirubin suppresses inflammatory responses in vivo, through the use of established rodent models of inflammation. Studies will specifically evaluate the ability of bilirubin to regulate NF-kappaB nuclear translocation, the expression of key NF-kappaB dependent pro-inflammatory enzymes, and the activation of proteinase-activated receptors (PARs). Specific Aim 2 will focus on investigating whether bilirubin exerts a direct effect on the activity of enzymes essential to the inflammatory response, including cyclooxygenases, nitric oxide synthase, phospholipases, and prototype serine protease activators of PARs (e.g., trypsin, thrombin). Kinetic and binding analyses will facilitate elucidation of the mechanisms underlying bilirubin's effects. Based on the findings of the above outlined analyses, future studies will focus on the mechanism(s) whereby bilirubin suppresses NF-kappaB activation and on the local regulation of bilirubin production and catabolism at sites of inflammation. It is anticipated that the results of these studies will provide a more comprehensive understanding of the role that bilirubin plays in the regulation of inflammation. As bilirubin is fairly innocuous in adults even at high concentrations, the findings of the proposed experiments may lay the foundation for potential new therapies for the treatment of inflammatory conditions.